This invention relates to novel soluble variants of the type I membrane proteins and to methods for making and using them. More specifically this invention relates to membrane proteins, and to methods of making and using them in the diagnosis and treatment of cancer.
The GA733-2 antigen has been found to be associated with a variety of human carcinomas such as colorectal, pancreatic, and breast carcinoma [D. Herlyn et al, J. Immunol. Meth., 73:157-167 (1984); H. G. Gottlinger et al, Int. J. Cancer, 38:47-53 (1986)].
GA733-2 is a 40 kDa human cell surface glycoprotein antigen that is associated with carcinomas of various origins. Its biological function remains unknown. Hydrophobicity analysis of the protein sequence predicted by cDNA has suggested that the GA733-2 antigen is a type I membrane protein, i.e., it possess signal peptide, extracellular domain, trans-membrane domain and intracellular anchor. An amino-terminal 23 residue signal peptide is followed by a 242 residue extracellular domain containing 12 cysteine residues and 3 potential N-glycosylation loci, a 23 residue trans-membrane domain, and a highly charged 26 residue intracellular anchor [S. Szala et al, Proc. Natl. Acad. Sci, (USA), 87:3542-3546 (1990); see also, M. S. Perez and L. E. Walker, Journal of Immunology, 142:3662-67 (1989); J. Strnad et al, Cancer Res., 49:314-17 (1989); and B. Simon et al, Proc. Natl. Acad. Sci. (USA), 87:2755-59 (1990].
GA733-2 is a monoclonal antibody (mAb) defined antigen [A. H. Ross et al, Biochem. Biophys. Res. Comm., 135:297-303 (1986)]. Several independently derived mAbs GA733, CO17-1A, M77, M79, 323/A3, among others, all define the GA733-2 antigen [See, for example, D. Herlyn et al, J. Immuno. Methods, 73:157-176 (1984), M. Herlyn et al, Proc. Natl. Acad. Sci. (USA), 75:1438-1482 (1979) and M. Herlyn et al, Hybridoma, 5:S3-S10 (1986) for discussion of CO17-1A; H. G. Gottlinger et al, supra, for discussing of M77 and M79; and D. P Edwards et al, Cancer Res., 48:1306-1317 (1986) for discussion of 323/A3].
Monoclonal antibodies that define tumor cell surface antigens are often being evaluated for the diagnosis and immunotherapy of cancer. Initial studies of mAbs CO17-1A and GA733 have demonstrated both cytotoxic effects in vitro and tumoricidal responses in vivo in experimental animal models. Clinical trials have shown strong mAb tumor binding [D. Herlyn et al, xe2x80x9cInitial Clinical Evaluation of Two Murine Monoclonal Antibodies for Immunotherapy of Gastrointestinal Carcinoma,xe2x80x9d Am. J. Clin. Oncol., (in press) (1991)]. Cases of partial and complete regression of disseminated cancer have also been reported [H. F. Sears et al, J. Biol. Resp. Mod., 3:138-150 (1984); and J. E. Frodin et al, Hybridoma, 7:309-321 (1988)]. Since only microgram quantities of the native antigen are available, therapeutic approaches to date have been limited to passive immunization with mAb and active immunization with anti-idiotype mAb (A2).
Molecular clones for the GA733-2 antigen have been isolated by immunoselection of COS cells transfected with a cDNA expression library derived from a human colon carcinoma cell line [S. Szala et al, Proc. Natl. Acad. Sci. (USA), 87:3542-3546 (1990)]. The GA733-2 sequence is identical to independently isolated cDNAs encoding the adenocarcinoma-associated antigen [J. Strand et al, Cancer Res., 49:314-317 (1989); and M. E. Perez and L. E. Walker, J. Immun., 142:3662-3667 (1989)]and the epithelial glycoprotein antigen [B. Simon et al, Proc. Natl. Acad. Sci. (USA), 87:2755-2759 (1990)]. The GA733-2 coding region is 54% identical to the GA733-1 gene, a retroposon that is abundantly transcribed in pancreatic carcinoma cell lines [A. J. Linnenbach et al, Proc. Natl. Acad. Sci. (USA), 86:27-31 (1989)]. The GA733-2 chromosomal gene contains exons encoding a epidermal growth factor-like repeat and an thyroglobulin type I repeat [A. J. Linnenbach, unpublished observation).
To date, other human tumor-associated antigens have been expressed in the vaccinia virus vector system: the epithelial tumor antigen expressed by breast carcinomas [M. Hareuveni et al, Proc. Natl. Acad. Sci. (USA), 87:9498-9502 (1990)]; and the melanoma-associated glycoprotein p97 [C. D. Estin et al, Proc. Nat. Acad. Sci. (USA), 85:1052-1056 (1988); and C. D. Estin et al, J. Natl. Cancer Inst., 81:445-448 (1989)].
Recombinant p97 antigen has induced specific humoral, cellular, and protective immunity in mice, and humoral and cellular immunity in monkeys. These observations emphasize the potential usefulness of a recombinant human tumor-associated antigen as vaccines for cancer patients.
Furthermore, the preparative isolation of secretable protein domains containing a specific region of interest (i.e., eptiopes) which can be used in biological, immunological, or physical (i.e., crystallography) assays, implies extended possibilities for studying the nature and function of other membrane proteins.
The baculovirus-insect cell expression system has been well recognized for its ability to abundantly express recombinant proteins which most often resemble native protein with respect to function, immunoreactivity, and immunogenicity. Baculovirus has been exploited for production of a variety of enzymes, trans-membrane proteins, and secretory proteins such as tissue plasminogen activator, interleukin-2, and human beta interferon. A soluble variant of the cell surface protein CD4 has been generated by expressing a restriction enzyme cleaved portion of the CD4 cDNA [R. E. Hussey et al, Nature (Lond.), 331:78-81 (1988)].
There remains a need in the art for an easily obtained and purified peptide which has the antigenicity of the GA733-2 membrane protein antigen in order to pursue immunological, physical and biochemical studies of this membrane protein, and to provide a means for diagnosis and immunotherapy against cancer.
In one aspect the present invention provides a polypeptide, designated GA733-2E. This polypeptide is a encoded by a truncated, modified version of native GA733-2 DNA, and permits secretion of an immunogenic fragment of the native GA733-2 antigen into the culture medium. This novel GA733-2E truncated antigen surprisingly retains the immunoreactivity and immunogenicity of the native full length GA733-2 antigen, and advantageously, may be easily purified from the culture medium into which it is secreted.
A further aspect of this invention is a pharmaceutical composition comprising GA733-2E as an active ingredient together with at least one substance selected from conventional pharmaceutical carriers, diluents, excipients and adjuvants. Optionally, GA733-2E may also be admixed with other active ingredients, including other GA733-2 variant peptides and other, known, cancer treating compounds. This composition may be used to elicit an immune response in a subject in a vaccine formulation. This composition is particularly useful in the treatment of various cancers.
Another aspect of this invention involves a method of eliciting a protective immune response in patients to certain tumors bearing a GA733-2 antigen by administering an effective amount of the a polypeptide and/or pharmaceutical composition described above. Such carcinomas include, but are not limited to, colorectal, pancreatic and breast carcinomas.
In yet another aspect, this invention provides diagnostic reagents, which include either the GA733-2E polypeptide or polynucleotide sequence, which sequence may be optionally associated with a detectable label, or bound to a solid support.
Thus still a further aspect of the invention are methods for diagnosing carcinomas which are characterized by the expression of native GA733-2antigen, which methods employ the recombinant GA733-2 polypeptide or polynucleotide sequences of the invention. Thus, the present invention provides a diagnostic kit for the detection of native GA733-2 antigen containing these diagnostic reagents.
Further, the invention provides the recombinant polypeptide GA733-2E, which may be used as a reagent for diagnostic purposes, and in methods for purifying and isolating the native antigen GA733-2.
In still another aspect, the invention provides a method of making soluble variants of GA733-2 proteins by omitting native GA733-2 DNA sequences for the trans-membrane and cytoplasmic domains, and creating a secretory protein from the extracellular domain. Preferably, this is accomplished via polymerase chain reaction (PCR).
Another aspect includes obtaining a soluble variant of any type I membrane protein produced by the method of omitting the DNA sequences for the transmembrane and cytoplasmic domains from the DNA sequence of said type I membrane protein. This truncated DNA sequence may then be produced by culturing a selected host cell transfect with a truncated DNA sequence in operative association with a regulatory sequence capable of directing the expression of the soluble variant. The soluble variant may also be produced by conventional synthesis.